MOLECULAR MECHANISMS OF DIET-INDUCED CARCINOGENESIS

流行病学和临床研究表明，人类叶酸缺乏可能导致对某些类型癌症的易感性。癌前异型增生可通过补充叶酸逆转。因此，阐明叶酸缺乏和癌症易感性的分子机制对于确定叶酸和其他饮食元素在癌症预防中的作用至关重要。一个良好的大鼠模型系统可用于研究低蛋氨酸、胆碱和叶酸饮食(亲脂饮食或LD饮食)在不含任何外源性异源物质的情况下诱导肝癌发生的作用。已知，在LD饮食诱发的肝癌中，一个关键的肿瘤抑制基因(P53)发生了甲基化。利用该模型系统，我们将(A)通过亚硫酸氢盐基因组测序和甲基化敏感的单核苷酸引物延伸(ms-Snupe)来研究肝癌发生不同阶段P53启动子上CpG二核苷酸的甲基化状态；(B)通过瞬时转染法研究特定CpG二核苷酸甲基化在P53启动子失活中的作用；(C)研究甲基化介导染色质结构改变的机制，并确定导致P53启动子沉默的关键因素。通过限制性内切酶可及性分析和针对甲基CpG结合蛋白(MeCPs)的染色质免疫沉淀(ChIP)技术(D)研究不同DNA甲基转移酶同工酶(参与维持和从头甲基化)在LD饮食诱导的肝癌发生过程中的表达和活性的调节；(E)鉴定与新甲基酶(F)相互作用的蛋白质；克隆这些基因，特别是RLGS技术检测到的在癌前病变肝脏中甲基化的三个基因，并对它们进行鉴定；研究它们在肿瘤发生的不同阶段的表达水平，并研究这些基因的沉默在肿瘤发生中的功能意义。叶酸/甲基缺乏与生长/肿瘤抑制基因或编码在肿瘤形成不同阶段抑制生长调控基因的蛋白质的区域高甲基化之间的关系，以及它们导致肿瘤形成的沉默，有望获得重要的信息。

Epidemiological and clinical studies have demonstrated that folate deficiency in humans could lead to susceptibility to certain types of cancers. Premalignant dysplasia could be reversed by folate supplementation. Elucidation of the molecular mechanisms underlying folate deficiency and predisposition to cancer is, therefore, of critical importance in determining the role of folate and other dietary elements in cancer prevention. An excellent rat model system is available to study the role of diet low in methionine, choline and folate (lipotrope-deficient or LD diet) on the induction of hepatocarcinogenesis in the absence of any exogenous xenobiotic agents. It is known that a key tumor suppressor (p53) gene is methylated in the hepatoma induced by LD diet. Using this model system, we will (a) explore by Bisulfite genomic sequencing and Ms-SNuPe (Methylation- sensitive single nucleotide primer extension) the methylation status of CpG dinucleotides on p53 promoter at different stages of hepatocarcinogenesis (b) investigate the role of methylation of specific CpG dinucleotides in p53 promoter inactivation by transient transfection assay (c) investigate the mechanism of methylation-mediated alteration in chromatin structure and identify the key factors involved in consequent silencing of p53 promoter with progression of tumorigenesis, by restriction endonuclease accessibility assay and chromatin immunoprecipitation (ChIP) with antibodies specific to methyl CpG binding proteins (MeCPs) (d) study the regulation of expression and activity of different DNA methyltransferase isozymes (involved in maintenance and de novo methylation) during hepatocarcinogenesis induced by LD diet (e) identify the proteins that interact with de novo methylases (f) clone the genes, specifically the three genes that are methylated in preneoplastic liver as detected by RLGS technique, identify them, investigate their expression levels at different stages of tumorigenesis and study functional significance of silencing of these genes in tumorigenesis. It is hoped that this study will yield important information concerning the relationship of folate/methyl deficiency to regional hypermethylation of growth/tumor suppressor genes or genes encoding proteins that suppress the growth regulatory genes at different stages of tumorigenesis, and their silencing that lead to tumor formation.